CRISPR/Cas9 targeted editing of UDP-rhamnose: Rhamnosyltransferase gene decreases its functions in acteoside biosynthesis and pest resistance in Rehmarmia glutinosa

UDP-rhamnose: rhamnosyltransferases (URTs)in Rehmarmia glutinosa (RgURT1-RgURT4)may catalyze two key downstream steps of acteoside biosynthesis. Moreover, they were identified from Rehmarmia glutinosa and preliminarily characterized, but their bioinformatics analysis and functions remain to be further explored. The present study mainly focused on investigating their bioinformatics function prediction, genotype-dependent expression, and roles for acteoside biosynthesis and pest resistance with CRISPR/Cas9 technology.Some key findings were as follows:they had a low identity but a typical PSPG box of rhamnosyltransferases, belonging to Glycosyltansferase-GTB type superfamily; They could be expressed depending on genotype,but RgURT4 expression is the highest; Based on RgURT4, two sgRNAs were designed and cloned into pBWA(V)HS-zmpl vector to construct a pBWA(V)HS-Cas9-RgURT vector. It was transferred to Rehmarmia glutinosa using Agrobacterium-mediated transformation so that hygromycin-resistant R. glutinosa plants were obtained. Sequencing indicated that CRISPR/Cas9 targeted editing resulted in base replacements in RgURT4,while its expression was decreased among these edited plants; A few of them had yellower leaves with white dots, lower acteoside and a little higher decaffeoylacteoside than WTs; Tetranychus cinnbarinus among them was observed by stereomicroscope. The results demonstrated that CRISPR/Cas9-mediated RgURT4 editing reduced the acteoside content and pest resistance but decaffeoylacteoside content of Rehmarmia glutinosa. This study will contribute to the function analyses of rhamnosyltransferases gene and downstream steps of acteoside biosynthesis as well as its CRISPR-Cas9-based molecular breeding.